Quaternary ammoniums and other preservatives' contribution in oxidative stress and apoptosis on Chang conjunctival cells

PURPOSE. To investigate some of the toxicity mechanisms of 10 preservatives currently used in ophthalmic solutions in vitro. METHODS. A continuous human conjunctival cell line was treated with differe nt concentrations of various preservatives for 15 minutes and for 15 minute s followed by 24 hours of cell recovery: three benzalkonium chlorides (BACs ) with different hydrocarbon chain length, benzododecinium bromide (BOB), c etrimide (Cet), phenylmercuric nitrate (PM), thimerosal (thi), methyl parah ydroxybenzoate (MPHB), chlorobutanol (clb), and EDTA. An inhibition study w as then conducted using a 1-hour vitamin E pretreatment followed by a 15-mi nute BAC treatment. Membrane integrity was assessed using a neutral red tes t and chromatin condensation with a Hoechst 33342 test. Reactive oxygen spe cies were measured using dichlorofluorescein diacetate test for H2O2 produc tion and hydroethidine test for O-2(-) production. These tests were perform ed using microplate cold light cytofluorometry. Cell size and DNA content w ere also analyzed using flow cytometry. Confocal microscopy was used to exp lore morphologic changes. RESULTS. A significant decrease of membrane integrity with chromatin conden sation was observed with all the quaternary ammoniums tested at concentrati ons of 0.005% and higher. The effect was amplified after 24 hours of cell r ecovery. The other preservatives tested did not decrease membrane integrity . H2O2 production was observed with all the preservatives, whereas O-2(-) p roduction was significantly higher with the quaternary ammoniums at 0.005% and 0.01%, compared with the other preservatives. Flow cytometry results co nfirmed the cytotoxicity observed with cold light cytofluorometry. CONCLUSIONS. The quaternary ammoniums tested (BAC, BOB, and Get) were the m ost cytotoxic preservatives in the current model. An apoptotic mechanism ap peared to be present at low concentrations of quaternary ammoniums, whereas a necrotic process appeared at higher concentrations. Superoxide anions ma y play an important role in tissue damage induced by preservatives in ocula r surface disorders.